Prostate cancer (PCa) is the most commonly diagnosed no-skin cancer in the US. With veterans comprised of 1/4th of our adult male population this cancer has huge impact on veteran health. Occupational exposures to exogenous and endogenous stresses further increase the risk and incidence in our veteran populations. Androgen-deprivation therapies (ADT) are the mainstay treatments for advanced prostate cancer (PCa). Yet, after a short remission, most PCas regrow in castration-level of androgen and become castration resistant PCa (CRPC). Recent approval of abiraterone (Abi), a CYP17A1 inhibitor, shows high efficacy in extending survival by blocking adrenal and intratumural androgen synthesis. Sadly, Abi-resistance inevitably emerges and patients are left with limited options. Thus, CRPC is in need of new therapies. We recently reported G-protein coupled estrogen receptor 1 (GPER1), the third estrogen receptor, is a potential druggable target for CRPC. It is upregulated in castration-resistant (CR) xenografts and in CRPC metastases specimens compared with levels in primary PCas. G-1 or (1(1-[4-(6-bromobenzo[1,3]dioxol-5-yl)-3a,4,5,9b- tetrahydro-3H-cyclopenta[c] quinolin-8-yl]-ethanone), a specific ligand for GPER1, is effective in inhibiting the growth of CR tumors but not those grown in intact hosts as GPER1 expression is markedly induced by castration. These findings suggest GPER1 is a novel therapy with specificity for CRPC. In addition to castration by itself, Abi-treated CR-xenografts showed increased expression of GPER1, indicating that one could potentiate the efficacy of G1 by co-treatments with Abi. In fact, we found the combinatorial treatment of CR tumors with G-1 and Abi showed synergistic efficacy, as Abi can upregulate GPER1 and release it from androgen receptor (AR)-mediated repression and in the presence of G-1, expression of several key androgen metabolic enzymes involved in Abi-resistance are significantly modulated. Based on these findings, we hypothesize that G-1 induced anti-PCa effect is mediated through GPER1, hence by maintaining its level either via conditional overexpression or complete androgen deprivation, we will enhance the therapeutic efficacy of G-1. Since GPER1 is an androgen repressed gene, we further hypothesize that AR-mediated inhibitory ncaRNA, DNA hypermethylation, and long range repression are mechanisms underlying GPER1 regulation. Two Aims are proposed to test these hypotheses: (1) Use conditional GPER1 ectopic expression and shRNA/GPER1 xenograft mouse models to manipulate the expression of GPER1 and maintain the G-1-induced tumor suppression effect. Additionally, we utilize Abi to induce GPER1 expression and test combinatorial G-1+Abi treatments for enhanced efficacy. We will identify the molecular pathways of action for Abi and G-1, individually and in combination, and decipher their common and differential resistance pathways. (2) Unravel the epigenetic mechanisms and long range chromatin interaction events responsible for mediating repression of GPER1 by androgen. Impact and significance: ADT is the current standard of care for CRPC patients before immuno- and chemo- therapies. Yet toxicity profiles and efficacies are of concern. The novelties of this proposal include studying (a) a non-androgen/androgen receptor signaling, novel drug G-1 which has higher efficacy and selectivity against CRPC; (b) the combinatorial treatments using G-1 and Abi to enhance each other's' efficacy; (c) the innovative mechanisms proposed for GPER1 regulation has transformative impacts in advancing our understanding of androgen repression of gene expression, a key to manage CRPC; (d) new knowledge on epigenetic regulation of GPER1 expression provides rationale for future combinatorial therapies with epigenetic drugs; (e) data from the ncaRNA and chromosome looping experiments are poised to generate new working hypotheses for revolutionizing concepts in androgen repression. Future probability of translation of this work to new treatment modalities for prolonging survival and improving quality of life in veteran PCa patients is high.